Analysis of penicillin-binding proteins ( PBPs ) in carbapenem resistant Acinetobacter baumannii

Gram-negative, cocco-bacillus which is known to cause nosocomial infections like septicaemia, bacteraemia, pneumonia, wound sepsis, endocarditis, meningitis, and urinary tract infections1-3. A. baumannii is resistant to desiccation and thus hard to eradicate once established in wards4,5. Carbapenem group, which includes imipenem and meropenem are last resort of β-lactams with highest efficacy and have Analysis of penicillin-binding proteins (PBPs) in carbapenem resistant Acinetobacter baumannii

broad spectrum against several Gram-negative bacteria including A. baumannii 6 .
Multidrug resistance in A. baumannii is a common clinical problem which further complicates the therapy [7][8][9] .The first known A. baumannii resistant to carbapenem was reported as early as in 1985 in Scotland 10 .The nosocomial outbreaks began in 1990s in all around the world and since then, incidence of carbapenem resistant isolates from hospitals worldwide has become a subject of concern [11][12][13][14] .In the past, multiple outbreaks of such infections caused by A. baumannii have been reported from different regions of India 7,15,16 .
Reports on resistance mechanisms in A. baumannii mainly focused on outer membrane impermeability 17,18 , production of beta lactamases 19,20 and production of efflux pump 21 .However, equally important for bacterial survival are penicillin-binding proteins (PBPs) which play a crucial role in the synthesis of peptidoglycan, an essential component of the bacterial cell wall 22 .PBPs catalyze the final step of polymerization (transglycosylation) and cross-linking by transpeptidation of peptidoglycan.PBPs can be one of the most likely mechanisms for carbapenem resistance.Direct association of alteration in PBPs and their properties with β-lactam resistance was reported in various bacteria like Escherichia coli K-12 23 , Pseudomonas aeruginosa 24 and Streptococcus pneumoniae 25 .It has been shown that modifications of PBPs play an important role in β-lactam resistance in Gram-negative bacteria 26 .Therefore, alterations in PBPs may play a role in drug resistance of A. baumannii.Although, the first carbapenem resistant strain of A. baumannii was reported more than 20 years back, till today there are just two reports, one from Spain 27 and one from Germany 28 which considered PBPs in resistance mechanism.Gehrlein et al 28 identified seven PBPs in a imipenem sensitive clinical isolate (from Germany) and imipenem resistant (I R ) clone of A. baumannii using radioactive 14 C-penicillin.In the imipenem resistant (I R ) clone of A. baumannii, they found a diminished expression of all the PBPs except 24 kDa PBP which is increased in its amount and showed low affinity for imipenem.Cuenca et al 27 from University Hospital in Spain found large variations in the PBP patterns (analyzed by 125 I Ampicillin reagent) and found only 6 PBPs (93, 84, 73, 64, 49 and 38 kDa) with the decreased expression of 73 kDa PBP in A. baumannii.Both the reports utilized the radioactive material for identification of PBPs.
In the present study, an attempt was made to identify PBPs of A. baumannii in ATCC and resistant strains isolates obtained in a tertiary care centre in north India, associated with carbapenem resistance.Materials: Bocillin FL was purchased from Molecular Probes.Inc., USA, Luria-Bertani (L-B) broth and agar were from Hi-media, India, Tris-HCl, EDTA, SDS and protein molecular weight standards from Bio-Rad laboratories USA.N-lauroyl sarcosine and β-mercaptoethanol and α-cyano-4-hydrocinnamic acid matrix were from Sigma Chemical Co., USA.Mass spectrometry grade trypsin was from Promega, USA.All other chemicals obtained from Merck, India were of analytical grade.GeBAflex-tube kit used for protein elution was from Gene Bio-Application, Israel.

Identification of penicillin binding proteins using
Bocillin FL: Total membrane protein extraction from A. baumannii was done with a slight modification in previous method 17,18 .Bacteria were grown in 250 ml Luria-Bertani broth at 37 0 C for 19.4 h (overnight and constant time period) and harvested by centrifugation at 5000 g for 15 min.Pellet was resuspended in potassium phosphate buffer (20 mM, pH 7.5) and cells were disrupted by ultrasonication under cold conditions.The suspension containing the cell envelope was treated with DNase and subjected to ultracentrifugation for 30 min at 100000 X g.The pellet (total membrane fraction) obtained was washed and resuspended in 1 ml phosphate buffer (≈1 mg/ml).Approximately, 40 µg of the total membrane protein fraction was incubated with 5 µM Bocillin FL at 37 0 C for one hour.Later, a 2 per cent N-lauroyl sarcosine was added to this reaction mixture and incubated for one hour.
Reaction mixture was subjected to ultracentrifugation (1, 00,000 X g) for 30 min and inner membrane fraction was separated as supernatant.Inner membrane protein fraction was then denatured with SDS-denaturing buffer at 100 0 C for 3-5 min; 20 µg of each reaction mixture was loaded on a 12 per cent SDS-PAGE.The protein gels were fixed using destaining solution immediately after electrophoresis and the protein bands were visualized under UV light (290 nm).The gels were photographed and the intensities were also measured using Bio-Rad gel Documentation.Isolation of proteins from each isolate was done three times and then the gel profiling was done for each extraction.Although it is well known that Bocillin binds selectively to PBPs only 31,32 , it was also checked whether Bocillin has any binding affinity with outer membrane proteins.To rule out this possibility, the total outer membrane extract was subjected with Bocillin and the resulting gel electrophoresis did not reveal any protein band confirming the fact that all those proteins detected by Bocillin in this study were only PBPs of inner membrane.
Protein elution: Penicillin binding protein (40 kDa) of ATCC 19606 was extracted using gel-elution technique.A 40 KDa protein from 12 per cent SDS PAGE of ATCC membrane was manually cut.The gel slice containing the protein was transferred to GeBAflextubes in a horizontal electrophoresis tank filled with protein PAGE running buffer and electroelution was carried out at 80 volts for two hours.The electroeluted protein sample was lyophilized and further dissolved in 100 µl of distilled water.Homogeneity of the sample was checked once again on SDS PAGE.

MALDI-MS analysis of purified PBPs:
Purified protein of apparent molecular weight 40 kDa was excised from the gel followed by in-gel trypsin digestion.The sample preparation for mass spectrometry was done according to Wilm et al 33 .Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis was performed on a Bruker Daltonics GmbH spectrometer (Germany).Analyses were carried out using α-cyano-4hydrocinnamic acid as a matrix.Monoisotopic peptide masses were assigned and database search was done using MASCOT v1.6b24 (Matrix Science, London, United Kingdom).The peptide mass tolerance was within 50 ppm.

Results & Discussion
Antimicrobial susceptibility and MIC: The antibiotic susceptibility test data revealed that all the 20 isolates of A. baumannii obtained from samples from ICUs, were resistant to a large number of antibiotics which include more than 60 per cent of β-lactams and related compounds.Only combination drugs of β-lactams with β-lactamase inhibitors showed a slightly higher potency in antimicrobial activity, similar to that reported in P. aeruginosa and A. baumannii 34 .A slightly higher potency of the combination drug (β-lactam + lactamase inhibitor) suggested that some lactamases were also present in the resistant strain.The resistance against β-lactams is a multigenic factor and thus we approached the problem by studying PBPs for the account of resistance in the clinical isolates.
The MIC levels for meropenem in all the specimens of A. baumannii ranged from 4 to >64 µg/ml.The resistant isolates could be broadly classified under 3 categories on the basis of their MIC for meropenem (Table I).The less resistant group (G1) included six isolates that showed an MIC level less than 16 µg/ ml.The intermediate resistant group (G2) included seven isolates with an MIC level ranging from 16 to 32 µg/ml and seven isolates of highly resistant group * MIC for meropenem: G1-≤ 8.0, G2-16.0-32.0,G3->32.0 (G3) showed MIC levels >32 µg/ml.It is known that penicillin has very little effect on the newly emerged resistant strains of A. baumannii.MIC of penicillin was given along with MIC of meropenem for comparison purpose only.
Inner membrane proteins (IMPs) profiling: Inner membrane consists of several proteins of signal transduction, electron transport system proteins, several transporters and PBPs, etc. PBPs are essentially located in the inner membrane; however, for detection of PBPs using Bocillin does not require inner membrane separation.To see how many of IMPs of A. baumannii are actually PBPs, the SDS PAGE profiles of IMPs were analysed (Fig. 1, lane 2).The total number of IMPs seen is quite large (>40 proteins).
Profiling of penicillin-binding proteins using Bocillin FL: Protein profile of PBPs of wild type ATCC 19606 of A. baumannii and 20 resistant isolates were obtained by SDS-PAGE.The penicillin-binding proteins are conventionally detected on SDS-PAGE using radioactive Benzyl-C 14 penicillin 23 .Detection methods using 125 I-labelled penicillin V and 3 H-or 14 Clabelled penicillin require days to week time 27 .The use of Bocillin FL offers several advantages over the radioisotope methods as it allows rapid detection up to 30 ng of a purified PBP under UV light and results can be obtained immediately after the SDS-PAGE.Detection of PBPs was done in a linear mode using 5 µM of Bocillin Fl which was much above the detectable concentration (0.8 µM) and much below the saturable concentration (25 µM) of Bocillin FL 31,32 .
PBPs in native strain: Before carrying out the PBP profiling on resistant isolates, the analysis on ATCC was done as a control.The analysis of the PBPs revealed eight bands in the wild type strain, which could be distinguished due to their well separated molecular weights (Fig. 1, lane 3).The eight PBPs had apparent molecular weights of 94 (PBP 1), 65 (PBP 2), 49 (PBP 3), 40(PBP 4), 30 (PBP 5), 24 (PBP 6), 22 (PBP 7), and 17 kDa (PBP 8).Gehrlein et al 28 showed the presence of only seven PBPs in a laboratory-sensitive clinical isolate of A. baumannii using radioactive 14 C-penicillin.In E. coli, a well documented Gram-negative bacteria; six PBPs in the range of 40-91 KDa were detected using Bocillin 31 .
Although one can see more than eight bands of PBPs, it is not possible to identify or resolve these bands separately.The relative expression levels of various PBPs in A. baumannii ATCC were quantitatively measured using densitometry.Of all eight PBPs, the PBP 4 (40 kDa) was highly expressed followed by the PBP 5 (30 kDa) and PBP 2 (65 kDa).The remaining PBPs were relatively less expressed.Although all the high molecular weight PBPs could be distinctly visualized under the UV light, PBP 6 and 7 with similar molecular weights (24 and 22 kDa, respectively) appeared as close bands and therefore were not clearly visualized on the gel.

Purification and mass spectrometry analysis of 40
KDa PBP: The major protein, expressed in the native strain of A. baumannii with apparent molecular weight of 40 kDa was purified using the GeBAflex-tube kit.The purity of the protein was checked on 12 per cent SDS-PAGE which gave a prominent single band at 40 kDa (Fig. 2).MALDI-TOF and peptide mass fingerprinting of purified protein is shown in Fig. 3.Of the 55 theoretical peptide masses obtained from mass spectrogram, 12 peptide masses on further analysis using MASCOT search showed identity with putative peptide sequences of PBP 2 (score 65%) of Clorobium tepidium (an anaerobic, green-sulphur bacterium) (Table II).CLASTALW 2.0.12 analysis of PBP proteins of A. baumannii and C. tepidium showed high homology (Alignment score 32).Moreover, protein BLAST search of the three identified peptides (peptide masses 1393.674,1789.845 and 1823.836) also showed the high homology with PBP2 protein (78, 88 and 71%) of A. baumannii.This further validated our according to their grouping based on their resistance towards meropenem.PBP profiling of all the 20 non repetitive isolates was done, and a representative profile of each group is presented in Fig. 4. The PBP in resistant isolates were grossly similar with minor variations present in their relative expression as compared to those of ATCC.For example, in group G1 isolates, the percentage of densitometric values of PBPs ranged between 30-63 per cent (PBP1); 33-63 per cent (PBP2); 68-83 per cent (PBP3); 77-91 per cent (PBP4); 66-76 per cent (PBP5); 86-98 per cent (PBP6); 49-69 per cent (PBP7); 95-99 per cent (PBP8) with reference to the respective PBP expression in ATCC.These types of variations in PBP expression in clinical isolates have been also reported by other workers 27 .
PBPs were clearly visualized in all the 6 resistant isolates of Group 1 (MIC <16 µg/ml).It was interesting to note that all 8 PBPS were downregulated as compared to those of ATCC.The most characteristic feature of G1 group was the absence of PBP 5 (30 kDa)  and simultaneous appearance of a new 28 kDa protein.
Most of the expression of 94 kDa was diminished.This suggests that PBPs of 94 and 30 kDa are adversely affected in the initial stages of resistance.
The banding pattern in Group 2 was quite similar to that of Group 1, there was further decrease in the intensities of PBPs (Fig. 4, lane 3).The expression levels of 94 kDa PBP (~10%), 24 kDa PBP (~20%) and 22 kDa PBP (~30%) were very low as compared to the native strain.
The third group (G3) consisted of resistant isolates with very high MIC of meropenem (~64 µg/ml).The PBP profiles of this group showed a pattern different   result that the purified 40 kDa protein was a penicillinbinding protein.

Comparative analysis of PBPs in resistant isolates:
Analysis of PBPs of resistant isolates was done from that of ATCC profile.In general, Group 3 showed just three obvious bands with molecular weight 65, 36 and 28 kDa.Similarly large variations in PBP profiles was also reported earlier with downregulation of 73 kDa as seen in 65 kDa PBP of our hospital strains 27 .After high antibiotic load in A. baumannii, Gehrlein et al showed hyperproduction of 24 kDa, which was not observed in the present study 27,28 .Group 3 resistant isolates also displayed markedly diminished expression of the PBPs 94, 49, 24 and 22 kDa, making these practically absent (Fig. 4, lane 4).Most significant change noticed in Group 3 was manifestation of a 36 kDa PBP which might be a new PBP or altered product of a high molecular weight PBP.It is possible that conformation of the PBP with 36 kDa may not be conducive to accommodate carbapenem in resistant A. baumannii.
The bacteria can develop β-lactam resistance by altering their PBPs.To do this, the organisms can decrease their affinity to, β-lactams, overproduce a critical PBP, or produce a new or altered PBP.It is interesting to study the properties of 28 and 36 kDa proteins in order to understand structure-function relationship in β-lactam resistance of A. baumannii.Our study showed an association between deregulation of PBPs and the resistance in A. baumannii.
-organisms and growth conditions: ATCC 19606 and 20 non repetitive β-lactam resistant isolates of A. baumannii were obtained from the Department of Microbiology, All India Institute of Medical Sciences (AIIMS), New Delhi, India during 2006 to 2008.Most of the clinical isolates were originated from ICUs of cardiology and neurology departments of AIIMS.All the clinical samples were inoculated on Mac Conkey agar at 37 0 C. Bacterial stocks were stored at -70 0 C in 60 per cent glycerol.

Fig. 3 .
Fig. 3. Peptide mass fingerprinting of 40 kDa protein of A. baumannii using Matrix-assisted laser desorption mass spectrometry (MALDI-MS).Peptide mass fingerprinting is done in positive mode (M+H + ).X axis represents the molecular weight/charge of the marked peaks.Intensity (y axis) is in arbitrary units.

Table I .
MICs (µg/ml) of meropenem and penicillin for clinical strains of A. baumannii

Table II .
Peptide sequences of PBP 40 kDa obtained from MALDI-PMF using MASCOT showing similarity with PBP2 of Clorobium tepidium